Method for Treating Systemic DNA Mutation Disease

ABSTRACT

A treatment for systemic DNA mutation diseases accompanied with development of somatic mosaicism and elevation of blood extracellular DNA and, more particularly, to a treatment of diabetes mellitus and atherosclerosis. The inventive method consist from introducing a treatment agent into a circulating blood system of a patient diagnosed with systemic DNA mutation diseases when said treatment agent destroys extracellular DNA in said blood of said patient and wherein said treatment agent used to destroy said extracellular DNA is a DNASE enzyme: said agent might be administered in doses and regimens which sufficient to decrease number average molecular weight of circulating extracellular blood DNA in the blood of said patient; 
     such decrease of number average molecular weight might be measured by gel electrophoresis of extracellular blood DNA fraction from the blood of said patient. A DNASE enzyme may be further applied in a dose and regime that provide a DNA hydrolytic activity measured in blood plasma exceeding 1.5 Kunitz units per 1 ml of blood plasma for more than 12 hours within a period of 24 hours.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation in Part of U.S. Ser. No. 10/564,609filed on 1 Jul. 2006, which claimed the benefit of PCT/RU2004/000260,filed 20 Jan. 2005, both of which are incorporated by reference as iffully rewritten herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to medical treatment of thesystemic DNA mutation diseases accompanied with development of somaticmosaicism and elevation of blood extracellular DNA and, moreparticularly, to a treatment of diabetes mellitus and atherosclerosis.

2. Description of the Related Art

Mosaicism refers to a mixture of cells of different genetic compositionin one individual. When DNA mutation is detectable in number, but notall somatic cells in one individual, it is called somatic mosaicism.Development of somatic mosaicism has been recently recognized asimportant mechanism of systemic DNA mutation diseases progression(Gottlieb B et al., Selection and mutation in the “new” genetics: anemerging hypothesis, Hum Genet. 2010 Mar; 127(5): 491-501.) Importanceof somatic mosaicism involving disease-causing mutations has beenreported for variety of monogenic (reviewed by Youssoufian H., NatureReviews Genetics 3, 748-758, October 2002) and more recently formultifactor DNA mutation diseases: cardiac rhythm disorders (M. H.Gollob et al., Somatic mutations in the connexin 40 gene (GJA5) inatrial fibrillation, N. Eng. J. Med. 354 (2006), pp. 2677-2688.);atherosclerosis (S. De Flora et al., Mutagenesis and cardiovasculardiseases. Molecular mechanisms, risk factors, and protective factors,Mutat. Res. 621 (2007), pp. 5-17), systemic vascular disorders (B.Gottlieb et al., BAK1 gene variation and abdominal aortic aneurysms,Hum. Mutat. 30 (2009), pp. 1043-1047); immune deficiencies (Wada T. etal., Somatic mosaicism in primary immune deficiencies, Curr Opin AllergyClin Immunol. 2008 December; 8(6): 510-4); Alzheimer disease (Beck J Aet al., Somatic and germline mosaicism in sporadic early-onsetAlzheimer's disease. Hum Mol Genet. 2004 Jun. 15; 13(12): 1219-24.);diabetes mellitus (Emma L. Edghill et al, Origin of de novo KCNJ11mutations and risk of neonatal diabetes for subsequent siblings. TheJournal of Clinical Endocrinology & Metabolism Vol. 92, No. 51773-1777).

According to current knowledge the systemic DNA mutation diseasesrepresent very distinct subsets of human pathology different in etiologyand pathogenesis and accordingly has fundamentally different, usuallypalliative treatment modalities—cholesterol lowering therapy foratherosclerosis (New Concepts and Paradigms in Cardiovascular Medicine:The Noninvasive Management of Coronary Artery Disease, K. Lance Gould,THE AMERICAN JOURNAL OF MEDICINE, Volume 104, Jun. 22, 1998, pp. 2-17)and insulin therapy or insulin sensitization therapy for diabetesmellitus (Pharmacological Management of Diabetes: Recent Progress andFuture Perspective in Daily Drug Treatment, Gerard Emilien et al.,Pharmacol. Ther. Vol. 81, No. 1, pp. 37-51, 1999).

More recently the gene therapy was recognized as potential tool fordisease specific intervention which may target the function of certainspecific disease involved genes and provide more efficient cure based onrepair of existing genetic defects in atherosclerosis (Ishisaki A, etal., Novel ideas of gene therapy for atherosclerosis: modulation ofcellular signal transduction of TGF-beta family. Curr Pharm Des. 2006;12(7): 877-86; Harris J D, et al. ApoE gene therapy to treathyperlipidemia and atherosclerosis. Curr Opin Mol Ther. 2006 August;8(4): 275-87; Hayden et al. Gene therapy method for reducing risk ofatherosclerosis, U.S. Pat. No. 6,784,162) and diabetes mellitus (G BParsons, Ectopic expression of glucagon-like peptide 1 for gene therapyof type II diabetes, Gene Therapy (2007) 14, 38-48; L. Chan, In vivogene therapy for diabetes mellitus, Trends in Molecular Medicine, Volume9, Issue 10, October 2003, Pages 430-435; M. During, Compositions forgene therapy of diabetes, EP1889914).

However no cure exists which may target the evolution of disease causingDNA mutations leading to development of somatic mosaicism. Accordingly,the development of new effective, non-toxic method that may suppress thedevelopment of somatic mosaicism and consequently be effective cure forsystemic DNA mutation disease is an extremely important task.

Circulating extracellular nucleic acids were discovered more than 60years ago (Anker P Circulating DNA in plasma or serum, Clin Chim Acta.2001 November; 313(1-2): 143-6). However until now elevated levels ofextracellular blood DNA in systemic DNA mutation diseases, and inparticular in atherosclerosis and diabetes mellitus were considered onlyas useful diagnostic and research tool (El Tarhouny S. A. et al.,Assessment of cell-free DNA with microvascular complication of type IIdiabetes mellitus, using PCR and ELISA. Nucleosides Nucleotides NucleicAcids. 2010 March; 29(3): 228-36; Langford M P et al., Plasma levels ofcell-free apoptotic DNA ladders and gamma-glutamyltranspeptidase (GGT)in diabetic children. Exp Biol Med (Maywood). 2007 October; 232(9):1160-9; Arnalich F. et al., Prognostic value of cell-free plasma DNA inpatients with cardiac arrest outside the hospital: an observationalcohort study, Critical Care 2010, 14; Arnalich F. Association ofcell-free plasma DNA with preoperative mortality in patients withsuspected acute mesenteric ischemia, Clinica Chimica Acta, in press;Zhong S, Presence of mitochondrial tRNA (Leu (UUR)) A to G 3243 mutationin DNA extracted from serum and plasma of patients with type 2 diabetesmellitus 2000 June; 53(6): 466-9.)

Circulating extracellular nucleic acids have never been considered aspotential therapeutic target in systemic DNA mutation diseases.Accordingly, no therapeutic method was developed which targetsextracellular blood DNA in systemic DNA mutation diseases. Thus it makesimpossible to take any technical solution as prototype.

As used in this application, the following terms are meant to have thefollowing corresponding definitions.

Deoxyribonuclease (DNASE) is any enzyme that catalyzes the hydrolyticcleavage of phosphodiester linkages in the DNA backbone.

Extracellular blood DNA number average molecular weight—the numberaverage molecular weight is a way of determining the molecular weight ofa polymer. The number average molecular weight is the ordinaryarithmetic mean or average of the molecular weights of the individualDNA macromolecules. It is determined by measuring the molecular weightof n polymer molecules, summing the weights, and dividing by n. Thenumber average molecular weight of extracellular blood DNA can bedetermined by gel electrophoresis. The shift of extracellular blood DNAbands to low-MW areas reflect decrease number average molecular weightand in fact reflects enzymatic cleavage of extracellular blood DNA.

DNA mutation disease refers to diseases where specific DNA mutation hasbeen identified as single leading cause (monogenic or single genedisorders) or multifactor disorders resulting from mutations in multiplegenes, often coupled with environmental causes.

Systemic disease is one that affects a number of organs and tissues, oraffects the body as a whole.

SUMMARY OF THE INVENTION

The object of this invention is to develop high-performance andlow-toxic method for treatment of systemic DNA mutation diseasesaccompanied with development of somatic mosaicism and elevation of bloodextracellular DNA and, more particularly, to a treatment of diabetesmellitus and atherosclerosis.

According to the invention this task is resolved by introducing atreatment agent into a circulating blood system of a patient diagnosedwith systemic DNA mutation disease when said treatment agent destroysextracellular DNA in said blood of said patient and wherein saidtreatment agent used to destroy said extracellular DNA is a DNASEenzyme. In one of preferred embodiments said agent must be administeredin doses and regimens which sufficient to decrease number averagemolecular weight of circulating extracellular blood DNA in the blood ofsaid patient; such decrease of number average molecular weight might bemeasured by gel electrophoresis of extracellular blood DNA fraction fromthe blood of said patient. In one of preferred embodiments the methodaccording the invention can be effectively applied for treatment ofdiabetes mellitus and atherosclerosis. A DNASE enzyme may be furtherapplied in a dose and regime that results in a DNA hydrolytic activitymeasured in blood plasma that exceeding 1.5 Kunitz units per 1 ml ofblood plasma for more than 12 hours within a period of 24 hours.

The present invention suggests that systemic DNA mutation disease can betreated by reducing of circulating extracellular blood DNA levels.

Development of systemic DNA mutation disease in humans is accompanied byquantitative and/or qualitative change of blood extracellular DNA.

There are no analysis of blood extracellular DNA spectrum and itsbiological role in systemic DNA mutation disease prior to thisinvention. A search of the prior art reveals no published dataconcerning an analysis of blood extracellular DNA spectrum in systemicDNA mutation disease performed by direct cloning and without use ofpolymerase chain reaction (PCR). PCR can pervert a pattern of bloodextracellular DNA because of specificity of primers used foramplification. There is no available knowledge about genetic repertoireof extracellular blood DNA in patients suffering from systemic DNAmutation disease and about biological role of extracellular blood DNA incourse of these diseases. Nothing is known about potential therapeuticvalue of extracellular blood DNA enzymatic destruction for treatment ofsystemic DNA mutation disease; so, taking into account all aforesaid,the invention complies with requirements of “novelty” criteria (N).

As the applicant established by direct cloning and sequencing ofextracellular blood DNA without PCR (Polymerase Chain Reaction), theextracellular blood DNA of patients with systemic DNA mutation diseasecontains the unique quantitative and qualitative repertoire of genes,which non-randomly represents human genome and contains genetic elementsinvolved in to the development of the disease. It was shown thatextracellular blood DNA might promote the development of somaticmosaicism and systemic DNA mutation disease.

It was established that enzymatic destruction of extracellular blood DNAby DNASE enzyme when applied in certain surprisingly high specific doseshas significant therapeutic effect on the course of systemic DNAmutation disease.

Aforesaid new characteristics of the claimed invention are based on newideas about mechanism of development of systemic DNA mutation disease.In this way the claimed method conformances to requirements of“invention step” criteria (IS).

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages and features of the present invention have been explainedby detailed description of embodiments with references to drawings:

FIG. 1: Extracellular blood DNA fraction from patient suffering fromsystemic atherosclerosis increases the expression of NF-kappa B proteinin cultured aortic endothelial cells (A); treatment of extracellularblood DNA with DNASE enzyme ameliorates this effect (B); and FIG. 2: A:Increased survival of NOD diabetic mice treated with different doses ofDNASE 1-evident increase in survival of mice treated with 500 mkg/kgDNASE 1; B: evident decrease of average molecular weight ofextracellular blood plasma DNA (as measured by electrophoresis) in bloodof NOD diabetic mice treated with 500 mkg/kg DNASE 1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventive method is realized as follows:

Materials and Methods:

The following agents, which destroy extracellular blood DNA, were used:bovine pancreatic DNASE (Sigma, specific activity 2 400 Kunitz units/mg;Samson-Med, specific activity 1 500 Kunitz units/mg), recombinant humanDNASE 1 (Gentech, specific activity 1000 U/mg).

Extracellular DNA from blood plasma was isolated as follows: freshplasma (no more than 3-4 hours after sampling) with anticoagulant(sodium citrate) was centrifuged on Ficoll-Plaque Plus(Amersham-Pharmacia) during 20 minutes at 1500 g. at room temperature. ½of plasma was detached, not affecting the rest of cells on the Ficollpillow, and further centrifuged at 10000 g. during 30 min for separationfrom cell fragments and debris. Supernatant was detached, withoutaffecting of the sediment, and was toped up to 1% of sarkosil, 50 MMtris-HCl, pH 7.6, 20 MM EDTA, 400 MM NaCl, and than mixed with equalvolume of phenol-chloroform(1:1) mixture. The prepared emulsion wasincubated during 2 hours at t=65° C., then phenol-chloroform mixture wasseparated by centrifuging (500 g during 20 minutes, room temperature).The procedure of deproteinization with phenol—chlorophorm mixture wasrepeated 3 times, and then the water phase was processed with chloroformand diethyl ether. Separation from organic solvents was made bycentrifugation at 5000 g during 15 minutes. Then equal volume ofizopropanol was added to resulting aqueous phase and the mixture wasincubated overnight at 0° C. After sedimentation the nucleic acids wereseparated by centrifugation at 10000 g during 30 minutes. The sedimentof nucleic acids was dissolved in of 10 MM tris-HCl buffer, pH 7, 6 with5 MM EDTA, and inflicted to the CsCl gradient (1M, 2.5M, 5.7M) intest-tube for rotor SW60Ti. The volume of DNA solution was 2 ml, volumeof each step of CsCl was 1 ml. Ultracentrifugation was conducted inL80-80 (Beckman) centrifuge during 3 hours at 250000 g. DNA wascollected from the surface of each gradient step into fractions. Thesefractions were dialyzed during 12 hours (t=4° C.) and pooled. Presenceof DNA in fractions was determined by agar electrophoresis and DNA wasvisualized by ethidium bromide staining. The amount of DNA wasdetermined with spectrophotometer (Beckman DU70) in cuvetts (100 mkl) atwavelength of 220-230 nm.

NOD mice were obtained from <<Pushcino>> animal breeding house.

Example 1 DNASE Treatment Suppresses the Development of SomaticMosaicism

Frequency of HPRT gene's mutations in blood T-lymphocytes was studied asthe model of development of somatic mosaicism in vivo. The human HPRTgene, mapped to chromosome Xq26, codes for a constitutively expressed,but non-essential, enzyme involved in purine metabolism. Mutantperipheral blood T-lymphocytes which do not express a functional HPRTgene product can be enumerated and clonally expanded by selective growthin the normally toxic purine analog 6-thioguanine in the presence ofspecific mitogens and growth factors. In normal, unexposed individualsthe frequency of 6-thioguanine resistant T-lymphocytes is typically 10⁻⁶to 10 ⁻⁵ (R. J. Albertini, J. A. Nicklas, J. P. O'Neill, S. H. Robison,In vivo somatic mutations in humans: measurement and analy-sis, Annu.Rev. Genet. 24 21990. 305-326.) Molecular analyses of the mutant, HPRTdeficient, clones have demonstrated that 85% of the gene inactivatingmutations observed in unexposed adults arises by localized HPRT genealterations—single base changes, small deletions or insertions and frameshift

Selective lymphocyte cloning was performed using peripheral blood of 8female patients with different forms of advanced cancer who got surgicalresection at Kostushko municipal Hospital (St.Petersburg) andimmunomodulation therapy at neoadjuvant setting (Neovir, 250 mg IM onceevery 2 days for 3 weeks). Following surgical resection 4 patients werefurther treated by IV infusions of bovine pancreatic DNASE (Samson)according the following schedule: 2000 mkg/kg×4 times daily for 21 day.Following completion of treatment the patients were assayed for HPRT (−)mutation in blood lymphocytes.

Mononuclear cells were isolated from the whole blood samples usingFicoll-Paquee (Becton Dickinson). Mitogenic stimulation of the separatedlymphocytes (1×10⁶/ml) was initiated with 1 mg/ml phytohemagglutinin(PHA) in RPMI 1640 media supplemented with penicillin (100 U/ml),streptomycin (100 mg/ml), 20% nutrient medium HL-1 and 5% BSA at 5% CO2at 37° C. for 24 h. Following wash the cells were then seeded in 96-wellround-bottomed plates at cell density of 2×10 ⁴

cells per well in selection medium to determine cloning efficiency. Thecells were plated in 200 ml of the RPMI medium containing 1 mg/ml6-thioguanine, 0.125 mg/ml PHA, 20% HL-1 and 5% BSA supplemented withinterleukin-2 (BD Biosciences, 10 BRMP units/mi). Four 96-well plateswere seeded for each patient. After 5 days of culture, the colonies onmutant selection plates were scored for growth using an invertedmicroscope. The results of selective T-lymphocyte cloning are presentedin the table below:

Total number of HPRT⁻ wells (growth Patient Treatment wells positive) KN P DNASE 384 7 P G P DNASE 384 12 B A I DNASE 384 2 F V V DNASE 384 11S L S NO 384 47 G A N NO 384 22 P M I NO 384 31 E N V NO 384 55

Thus, inventive treatment suppresses spread of HPRT (−) mutation andsuppresses the development of somatic mosaicism.

Example 2 Extracellular Blood DNA Promotes the Development of SomaticMosaicism

The extracellular blood plasma DNA was purified from blood of patientENV as specified in methods section. Mononuclear cells were isolatedfrom the whole blood samples of patients KNP, PGP, BAI and FW asspecified in Example 1. The mitogenic stimulation and selective cloningwere performed as specified in Example 1 with modification as follows:during mitogenic activation stage lymphocyte cultures of patients KNP,PGP, BAI and FW were supplemented with 50 mkg/ml of extracellular bloodplasma DNA purified from patient ENV. After 5 days of culture, thecolonies on mutant selection plates were scored for growth using aninverted microscope. The results of selective T-lymphocyte cloning arepresented in the table below:

Total number of HPRT⁻ wells (growth Patient wells positive) K N P 384 18P G P 384 15 B A I 384 21 F V V 384 31

Thus, extracellular blood DNA promotes the development of somaticmosaicism.

Example 3 Sequencing of Extracellular Blood DNA from the PatientSuffering from Type 2 Diabetes and Systemic Atherosclerosis. Treatmentof Atherosclerosis

A 54-years-old man has been admitted to the Cardiothoracic surgerydepartment of Kostushko municipal hospital (St.Petersburg) in severecondition complaining on intensive pain in abdomen, diarrhea, intensivepain in legs that appear during walking, loss of weight. Diabetesmellitus type 2 was diagnosed 12 years ago and glybencamide wasprescribed. Pain in epigastrium after food intake appeared 15 monthsago. Antacids were prescribed but pain continued to increase andsteatorrhea appeared in the last 3 months. Because of intensive painsyndrome anorexia has developed in a couple of days prior admittance.Considerable exhaustion (body weight was 44 kg; body weight loss was 28kg for the last 5 months) and absence of arterial pulsation on legs werefound out during examination. No organic changes were observed duringgastroduodenoscopy and colonoscopy. Electrocardiographic data was notchanged pathologically. Moderate increase of cholesterol level andlow-density lipoprotein fraction was observed in blood analysis.Glycated hemoglobin' level was 11%. Partial occlusion of aorta belowrenal artery (70%), partial occlusion of iliac arteries (90%), totalocclusion of upper and lower mesenteric artery were observed onaortography.

The probes of patient's extracellular blood DNA were taken beforeinitiation and on day 35 of therapy. The extracellular DNA was cloned bythe method which allows to get non amplified plasmid libraries of bloodextracellular DNA with representativeness up to one million of cloneswith the average size of 300-500 base pairs. The DNA which has beenisolated using the protocol specified in Materials and Methods sectionwas treated with Proteinase K (Sigma) at 65° C. and subjected toadditional phenol-chloroform extraction step with further overnightprecipitation by ethanol. The DNA fraction was than treated by Eco RIrestrictase or by Pfu polymerase (Stratagene) in presence of 300 mkM ofall desoxynucleotidetriphosphates for sticky-ends elimination. Thecompleted DNA was phosphorylated by polynucleotidkinase T4 and ligatedto pBluescript plasmid (Stratagene), which had been digested with EcoRIor PvulI and dephosphorylated by phosphatase CIP (Fermentas). Theprocess of ligation was conducted with Rapid Legation Kit (Roche). Theligated library was transformed into DH12S cells (Life Technologies) byelectroporation (E. Coli porator; BioRad). 12-20 electroporation covetswere used for the transformation of one library. The library serialdilutions were cloned on 1.5% agar and LB media supplemented withampicillin. In both cases the libraries represented 2-3×10⁶ clones.

Analysis of 75 randomly selected clones with the size 300-1000 basepairs from the “before treatment” library revealed 56 clones containingunique human DNA sequences as presented at the table below:

Potential role in Number of atherosclerosis/diabetes Gene clonesmellitus Neutral endopeptidase 2 At atherosclerosis its activity isincreased in endothelial cells, nonstriated muscle cells, stromal cellsof artery' intima. Decreasing of its activity can decrease lipidsaccumulation in vessels wall. Muskelin 1 1 Works as mediator of cellresponse on thrombospondin 1. Thrombospondin 1 - mediated processes arepathophysiological components of atherosclerotic affection of arterywall. Nf-kappaB 3 At hyperglycemia and atherosclerosis activity isincreased in cells of artery wall. E-selectin 3 High level of expressionis a risk factor of angiopathy development at diabetes type 2. GAD2:glutamate 2 One of the main pancreatic decarboxylase 2 autoantigens.Phospholipase C, 2 Induces expression of epsilon receptors oflow-density lipoproteins. BAI3: brain-specific 1 Angiogenesis inhibitorangiogenesis inhibitor Nicotinamide 1 Involved in detoxification ofnucleotide reactive oxygen species and transhydrogenase insulinsecretion. 17 kD fetal brain 1 UNCLEAR protein CRTL1: cartilage 1Involved in morphogenetic linking protein 1 process in heart and largevessels Transient receptor 1 UNCLEAR potential cation channelThus, extracellular blood DNA from patient having diabetes mellitus andsystemic atherosclerosis contains significant non-random presence ofhuman disease-relevant unique genomic DNA.

Patient was considered as not eligible for surgery so, conservativetherapy was chosen. Intensive IV nutrition was started. Insulin andanti-aggregation therapy have being started. Under patient consent dailyintravenous infusions of bovine pancreatic DNASE (Samson) at daily doseof 800 mg (1 200 000 Kuntz units) divided to 4 two-hour deliveries werestarted. Week after start of DNASE therapy pain syndrome disappeared andpatient was allowed to take light dietetic food orally. 20 days afterstart of DNASE treatment patient was switched to full value oralnutrition. General state was improved, body weight has increased. 45days following initiation of DNASE treatment patient was reexamined byangiography. 20% decrease of aorta occlusion and 35% decrease of iliacartery occlusion level as well as appearance of blood circulation inupper and lower mesenteric was observed. Patient was considered aseligible for revascularization surgery.

Extracellular blood plasma DNA sampled from patients blood at day 35following start of DNASE therapy was assayed by gel electrophoresis andcloning. Analysis of 50 clones randomly chosen from the library obtainedfrom the extracellular blood plasma DNA of patient on the day 35 afterthe beginning of treatment has shown that more than 90% of revealedclone sequences are short fragments of repetitive human DNA with thedominance of alpha-satellite DNA.

Example 4 Influence of Extracellular Blood DNA from the Patient withSystemic Atherosclerosis on Disease Causing Protein Expression in AorticEndothelial Cells

Endothelial NF-kappa B signaling orchestrates proinflammatory geneexpression at the arterial wall and promotes the pathogenesis ofatherosclerosis.

Here we assayed the influence of extracellular blood DNA from thepatient diagnosed with systemic atherosclerosis on NF kappa B expressionin primary aorta endothelial cell culture. Blood plasma was obtainedfrom vascular surgery clinic of St.Petersburg Medical Academy from thepatient undergoing femoro-femoral bypass surgery due to severeatherosclerotic arterial occlusion.

The extracellular blood DNA was purified as described in Materials andMethods section. The aortic endothelial cells (C-006-5C; Invitogen) wereplated at density of 5-8×10² cells/mm² in multiwell (12×) cell cultureplates in Clonetics® EGM®-2MV media (Lonza Cologne AG) and incubated for48 h. at 37° C. and 5% CO₂. Following 24 h of growth the culture mediawas supplemented with 50 mkg/ml of patient extracellular blood DNAfraction or 50 mkg/ml of patient extracellular blood DNA fraction plushuman recombinant DNASE-1 (Genentech) at 1 mkg/ml concentration.

After 24 h. culturing the explants were lysed in buffer containing 20 mMTris-HCl, 150 mM NaCl, 1 mM phenylmethylsulfonylfluoride, 5 mg/mlaprotinin, 0.5% Nonidet P-40 (Sigma-Aldrich) for 1 hour at 4° C. Thelysates were centrifuged for 10 min at 20,000 rpm. The supernatants werediluted with reducing sample buffer and were separated byelectrophoresis on a 10% SDS-PAGE gel (20 mkg protein per lane loaded).The proteins were transferred onto Hybond-C-nitrocellulose membrane(Amersham Italia, Milan, Italy. For immunoblot analysis, the membraneswere incubated with the NF-κB antibodies (Stressgen). The bands weredetected using the chemiluminescence system.

The results are presented at the FIG. 1. Extracellular blood DNAfraction from patient suffering from systemic atherosclerosis increasesthe expression of NF-kappa B in cultured aortic endothelial cells andtreatment with DNASE ameliorate this effect.

Example 5 Treatment of Diabetes Mellitus

Non-obese diabetic (NOD) mice exhibit a susceptibility to spontaneousdevelopment of autoimmune insulin dependent diabetes mellitus. 60 NODmice were recruited to the study at the age of 14 weeks when all of thembecame hyperglycemic. The recombinant human DNASE 1 (Gentech) at 50mkg/kg and 500 mkg/kg was administered intramuscularly twice daily for21 day. 2 mice from each group were sacrificed at the last treatment dayto perform evaluation of extracellular blood plasma DNA. The efficaciesof DNASE were assessed based on the survival rate at day 35. The resultsof experiments are presented at the FIG. 2. There is evident increase insurvival of mice treated with 500 mkg/kg DNASE 1; such survival isaccompanied with decrease of average molecular weight of extracellularblood plasma DNA (as measured by electrophoresis) in blood of NODdiabetic mice. Thus, high doses of DNASE according to inventivetreatment are able to decrease the quantities of circulatingextracellular blood plasma DNA and are effective against systemic DNAmutation disease-diabetes mellitus.

Example 6 Treatment of Diabetes Mellitus

A 46-years-old patient with 3 years history of type 2 diabetes mellituswas admitted to the internal therapy clinic of St.Petersburg MedicalAcademy. Patient failed to achieve proper glucose control using oralhypoglycemic agents including those of thiazolidinediones,biguanides_and sulfonylureas. Patient was switched to 0.3 IU/kg of NPHinsulin monotherapy (21 IU daily) and discharged from clinics under thesupervision of ambulatory endocrinologist. Three month later patient wasreadmitted to the clinic since glycosylated hemoglobin (HbA1) level wasstill too high (above 10%) with evolving microalbuminuria and decreasein vision sharpness despite daily insulin dose was adjusted up to 1.2U/kg (84 U/day) during ambulatory period. Under patient consent he wasassigned for intramuscular injections of bovine pancreatic DNASE(Samson) twice daily at 200 mg/day dose for 4 months and againdischarged from clinics. At 4 month after initiation of treatmentpatient were reexamined in clinics outpatient department. Significantimprovement in insulin sensitivity, improvement of glycemia control andnormalization of kidney function has been reported by patient ambulatoryendocrinologist and confirmed by laboratory examination in clinic. Theeffect of DNASE treatment on patient disease indicators is presented intable below:

At DNASE course Indicator Prior DNASE treatment completion Insulinrequirement 1.2 IU/kg 0.6 IU/kg HbA1 13.2% 7.2% 24 h. albuminuria 275 mg60 mgThus, the inventive treatment is effective in diabetes mellitus.

INDUSTRIAL APPLICABILITY

For the realization the methods there were used well-known materials andequipment manufactured in plant conditions and according to aforesaidthe invention conformances to requirements of “industrial applicability”criteria (IA)

1. A method for treating of systemic DNA mutation disease accompaniedwith development of somatic mosaicism and elevation of bloodextracellular DNA level said method comprises a step of introducing atreatment agent into a circulating blood system of a patient diagnosedwith systemic DNA mutation disease said treatment agent destroysextracellular DNA in said blood of said patient, wherein said treatmentagent used to destroy said extracellular DNA is a DNASE enzyme.
 2. Amethod according claim 1 wherein DNASE enzyme is administered in dosesand regimens which sufficient to decrease number average molecularweight of circulating extracellular blood DNA in the blood of saidpatient which decrease as measured by gel electrophoresis ofextracellular blood DNA fraction from the blood of said patient.
 3. Amethod according claim 1 where systemic disease is diabetes mellitus. 4.A method according claim 1 where systemic disease is atherosclerosis. 5.A method according claim 1 when DNASE enzyme is applied in a dose andregime that results in a DNA hydrolytic activity measured in bloodplasma that exceeding 1.5 Kunitz units per 1 ml of blood plasma for morethan 12 hours within a period of 24 hours.